Methods of sequencing DNA are typically performed by either the chemical degradation method of Maxam and Gilbert (Methods in Enzymol. 65:499-560 (1980)) or the enzymatic dideoxynucleotide termination method of Sanger et al. (Proc. Natl. Acad. Sci. U.S.A. 74:5463-67 (1977)). In the chemical method, base specific modifications result in a base specific cleavage of the radioactive or fluorescently labeled DNA fragment. With the four separate base specific cleavage reactions, four sets of nested fragments are produced which are separated according to length by polyacrylamide gel electrophoresis (PAGE). After autoradiography, the sequence can be read directly since each band (fragment) in the gel originates from a base specific cleavage event. Thus, the fragment lengths in the four "ladders" directly translate into a specific position in the DNA sequence.
In the enzymatic chain termination method, the four base specific sets of DNA fragments are formed by starting with a primer/template system elongating the primer into the unknown DNA sequence area and thereby copying the template and synthesizing a complementary strand using DNA polymerases, such as Klenow fragment of E. coli DNA polymerase I, a DNA polymerase from Thermus aquaticus, Taq DNA polymerase, or a modified T7 DNA polymerase, Sequenase (e.g., Tabor et al., (1987) Proc. Natl. Acad. Sci. U.S.A. 84:4767-4771), in the presence of chain-terminating reagents. Here, the chain-terminating event is achieved by incorporating into the four separate reaction mixtures in addition to the four normal deoxynucleoside triphosphates, dATP, dGTP, dTTP and dCTP, only one of the chain-terminating dideoxynucleoside triphosphates, ddATP, ddGTP, ddTTP or ddCTP, respectively, in a limiting small concentration. The four sets of resulting fragments produce, after electrophoresis, four base specific ladders from which the DNA sequence can be determined. Undesirably, most methods for sequencing DNA require the use of polyacrylamide gel electrophoresis (i.e., PAGE) that can result in sequencing artifacts or require detectable labels, such as radioisotopes, enzymes, or fluorescent or chemiluminescent moieties.
Using DNA sequencing methodologies, the entire sequence of the human genome will be determined. The knowledge of the complete sequence of the human genome DNA will certainly help to understand, to diagnose, to prevent and to treat human diseases. To be able to tackle successfully the determination of the approximately 3 billion base pairs of the human genome in a reasonable time frame and in an economical way, rapid, reliable, sensitive and inexpensive methods needed to be and still need to be developed.
Therefore it is an object herein to provide additional methods for sequencing. In particular, it is an object herein to provide mass spectrometric methods of sequencing nucleic acids using RNA polymerase. It is a further object herein to provide methods of sequencing nucleic acids in an array format using RNA polymerase in which nucleic acid probes are immobilized to supports at high densities to facilitate mass spectrometric detection. It is also an object herein to provide methods for identifying transcriptional terminator sequences using mass spectrometric methods.